1. Field of the Invention
The invention relates to methods for modifying the surfaces of ceramic materials in order to alter the interfacial energy of these surfaces with liquids, thereby rendering the ceramic materials more suitable for use as sample holders for electrophoresis and/or isoelectric focusing. The invention also relates to devices made with ceramics having such modified surfaces, and in particular relates to ceramic vessels used in electrophoresis.
2. Description of Related Art
The ability of a liquid phase to wet a solid phase is related to the difference between the work of adhesion (i.e., the work required to separate the immiscible liquid and solid phases) and the work of cohesion (i.e., the work required to separate the liquid from itself). If the work of adhesion is sufficiently greater than the work of cohesion, the liquid-solid system will have a positive spreading coefficient, and wetting of the solid by the liquid will spontaneously occur. If the work of cohesion of the liquid is greater than the work of adhesion, the spreading coefficient will be negative, and wetting will not spontaneously occur (because additional work will be required to overcome the attraction of the liquid for itself and make it spread across the solid surface). The determination of works of adhesion, works of cohesion, and spreading coefficient are related to the surface tension, and to the closely related concept of contact angle.
Surface tension can be thought of as the change in Gibbs free energy per unit change in the surface area. Contact angle is measured at a gas, solid, liquid interface of a sessile or pendant drop of liquid on a solid surface, typically by an optical comparator. A larger contact angle, xcex8, indicates a decreased wetting by the liquid of the solid. A contact angle of 0 indicates that the liquid completely wets the solid. See Hiemenz, Principles of Colloid and Surface Chemistry, Marcel Dekker, 1977, pp. 209-251, the entire contents of which is hereby incorporated by reference.
Isoelectric focusing (IEF) is a technique widely used to separate proteins according to their different isoelectric points. A sample containing proteins to be separated is placed on a gel, often a single lane gel or gel strip having a pH gradient (such gels are typically obtained by electrophorescing carrier ampholytes through the gel or by covalently incorporating a gradient of acidic and basic buffering groups when the gel strip is cast). The protein molecules migrate along the gel in response to an applied electric field until they reach a point in the gel where the gel pH matches the protein""s isoelectric point (i.e., the pH at which the net charge on the protein is zero). Isoelectric focusing can be used to discriminate between proteins having differences in isoelectric point as small as 0.01. See Stryer, Biochemistry, 4th. ed., pp. 46-48, the entire contents of which are hereby incorporated by reference.
In order to increase resolution of the isoelectric focusing, it is desirable that the proteins be denatured prior to and during separation. Denaturation helps to provide a single protein configuration for each protein, and to minimize interactions between protein molecules or aggregation, as well as to expose internal ionizable amino acids. Denaturation, as well as solubilization of the protein, is typically achieved by placing the protein in a solution containing urea and/or detergent prior to application to the gel.
In SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), mixtures of proteins are separated according to the difference in protein molecular weights. The protein is contacted with SDS, which is an anionic detergent. The detergent both denatures the protein, and provides a large negative charge to the protein molecules, swamping the effect of any charge carrying groups on the protein itself, and providing a mechanism by which the protein will migrate in an electric field. The protein mixture is typically combined with SDS or applied to a gel containing SDS, and electrophoresed down the gel, so that proteins having lower molecular weights travel farther.
Two-dimensional (or 2-D) electrophoresis is a widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. The technique sorts proteins by combining the IEF and SDS-PAGE techniques in two discrete steps. In one of these steps, generally the first step, IEF is used to separate the proteins according to their differing isoelectric points. The other, generally second, step separates the proteins according to their molecular weight, using SDS-PAGE gel electrophoresis. The molecular weight separation is carried out across a dimension of the gel normal to the first dimension of the gel (i.e., normal to the pH gradient). Typically, this is done by placing the strip obtained from IEF across the top of a polyacrylamide gel containing SDS and applying an electric field. The result is a two-dimensional xe2x80x9cmapxe2x80x9d of spots of separated proteins, each having a characteristic pI and molecular weight. With a large enough gel, 2-D electrophoresis can be used to separate large numbers of different proteins from a single sample. In addition to providing information about the isoelectric points, and apparent molecular weights of these proteins, the amount of protein present in the sample may also be determined. 2D electrophoresis is also useful to analyze cell differentiation, detect disease markers, monitor therapies, micropurify proteins, as well as in cancer research and drug discovery.
The gels used for isoelectric focusing and for 2-D electrophoresis can be supplied in the form of prepared strips that are then supported by a stripholder. Solution to rehydrate the strips and/or apply the sample thereto may be supplied to the stripholder, and the strip inserted.
Ceramic materials, while desirable for use in electrophoresis equipment due to their high electrical breakdown strength, high thermal conductivity, chemical inertness, and low cost, can create problems in such applications due to the somewhat hydrophilic nature of the ceramic surfaces. In particular, when used to make stripholders of the type described above, the urea-containing and/or detergent containing protein carrying solutions tend to wick over the stripholder walls. The wicking solutions carry protein sample with them, leading to loss of sample material and potentially inaccurate results of the isoelectric focusing and 2-D electrophoresis.
Similar problems occur with ceramic materials used for microarrays of multiwell plates used in combinatorial chemistry.
Accordingly, there is a need in the art for methods of treating ceramic surfaces to lessen or avoid the wicking phenomenon responsible for sample loss and potentially inaccurate electrophoresis results, and for ceramic surfaces so treated and for articles, in particular IEF and electrophoresis sample holders, made therefrom.
The invention provides two methods for modification of ceramic surfaces so as to reduce or avoid the wicking phenomenon associated with sample loss and potential inaccuracies in IEF or electrophoresis sample holders.
In one method, the surface of the ceramic material that will come into contact with aqueous solutions is mechanically polished with an abrasive material until the wettability of the ceramic surface by the aqueous solution is decreased. This decrease is sufficient to lessen or prevent wicking. In a particular embodiment of this method, the ceramic surface is the surface of a IEF or gel electrophoresis sample holder or a microarray plate for use in combinatorial chemistry.
In another method, the surface of the ceramic material is modified by a silane heat-treatment method, whereby a silane is contacted with the ceramic surface and heated for a sufficient time and at a sufficient temperature to decrease the wettability of the ceramic surface (and, it is believed, the silane is covalently reacted with the hydroxyl moieties on the ceramic surface). In a particular embodiment of this method, the ceramic surface is the surface of a IEF or gel electrophoresis sample holder or a multiwell microarray plate for use in combinatorial chemistry. Desirably, these sample holders have a ceramic surface having a contact angle of greater than about 40xc2x0 when in contact with an aqueous solution of from about 6 M to about 9.8 M urea.
Both methods are believed to modify the surface characteristics of the ceramic material in such as way as to decrease its ability to adhere to aqueous solutions, including aqueous solutions of biological molecules, such as urea-containing or detergent-containing electrophoresis solutions. This decreased adhesion (i.e., increased hydrophobicity) between the ceramic material and the aqueous solutions leads to decreased wettability, as measured by an increase in contact angle. The resulting ceramic material is suitable for use in articles requiring decreased wettability, and in particular, for gel or gel strip holders used in IEF or 2-D electrophoresis and microarrays or microwell plates of the type used in combinatorial chemistry. When used in these applications, the ceramic material prepared by the invention lessens or avoids the loss of sample material that has resulted from wicking of sample solutions out of the ceramic container. This avoidance of sample material loss increases the accuracy and reliability of the IEF, SDS-PAGE, combinatorial, or other analytical procedures carried out on the sample.